Protein purification and characterization: The partially purified uridylyl removing/uridylytransferase enzymes(s) have been sized at 140,000 Daltons. A nucleotide monophosphate binding substance from E. coli was enriched 50-60 fold by precipitation with streptomycin sulfate and ethanol fractionation. A six fold purification of PII regulatory protein, already 200 fold enriched, was achieved by passage over a mercury-agarose column. PII survived exposure to 2-6 M urea. Steady stare analysis of the deadenylylation reation suggests a reaction scheme in which ATP and alpha-ketoglutarate bind to adenyly transferase independently, each enhancing the binding of the other. The binding sites of glutamine and alpha-KG on adenylyl transferase appear to be separate. Inhibition of uridylyl removing activity by a variety of compounds such as coenzyme A and CMP is non-competitive with respect to substrate (PIID). The effects of these inhibitors and the enhancement of uridylyl removing activity by glutamine are very sensitive to pH. Colorometric assays for uridylyl transferase and uridylyl removing activities have been adapted to an assay developed by S. Rhee for the purpose of measuring PIID.